The long journey towards standards for engineering biosystems: Are the Molecular Biology and the Biotech communities ready to standardise?

Synthetic biology needs to adopt sound scientific and industry-like standards in order to achieve its ambitious goals of efficient and accurate engineering of biological systems.

A minimum information standard for reproducing bench-scale bacterial cell growth and productivity.

Reproducing, exchanging, comparing, and building on each other's work is foundational to technological advances. Advancing biotechnology calls for reliable reuse of engineered organisms. Reliable reuse of engineered organisms requires reproducible growth and productivity. Here, we identify the experimental factors that have the greatest effect on the growth and productivity of our engineered organisms in order to demonstrate reproducibility for biotechnology. We present a draft of a Minimum Information Standard for Engineered Organism Experiments (MIEO) based on this method. We evaluate the effect of 22 factors on Escherichia coli engineered to produce the small molecule lycopene, and 18 factors on E. coli engineered to produce red fluorescent protein. Container geometry and shaking have the greatest effect on product titer and yield. We reproduce our results under two different conditions of reproducibility: conditions of use (different fractional factorial experiments), and time (48 biological replicates performed on 12 different days over 4 months).

Measurements of translation initiation from all 64 codons in E. coli.

Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the 'start codons' for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.

Relation of concavity in the expiratory flow-volume loop to dynamic hyperinflation during exercise in COPD.

Active expiration during exercise can increase intrathoracic pressure yielding concavity in the expiratory flow-volume loop in COPD. We investigated the relationship between this concavity and dynamic hyperinflation (DH). 17 COPD patients (FEV1: 38±10%pred, GOLD stage 3-4) and 12 healthy subjects performed cycle ergometer incremental exercise. Expiratory limb of the spontaneous flow-volume loop was analyzed breath-by-breath using a geometric approach (rectangular area ratio (RAR), Respir. Med., 104(3):389-96, 2010). RAR below 0.5 demonstrates expiratory limb concavity. DH was determined with serial inspiratory capacity maneuvers. 5 of 17 patients displayed little end-exercise concavity (RAR=0.52±0.04, group LCONC). 12 patients had concavity at rest and end-exercise RAR reached 0.40±0.03 (group HCONC). Healthy subjects showed no concavity. End-exercise RAR correlated with resting FEV1%pred (R2=0.81, P<0.05). Group HCONC, compared to groups LCONC and H, reached significantly lower work rate, minute ventilation, and more dyspnea. DH inversely correlated with RAR (R2=0.81, P<0.05). Detection of concavity in spontaneous flow-volume loops may help assess DH and exercise limitation in COPD.

When Wavelengths Collide: Bias in Cell Abundance Measurements Due to Expressed Fluorescent Proteins.

The abundance of bacteria in liquid culture is commonly inferred by measuring optical density at 600 nm. Red fluorescent proteins (RFPs) can strongly absorb light at 600 nm. Increasing RFP expression can falsely inflate apparent cell density and lead to underestimations of mean per-cell fluorescence by up to 10%. Measuring optical density at 700 nm would allow estimation of cell abundance unaffected by the presence of nearly all fluorescent proteins.

Fractal dimension of microbead assemblies used for protein detection.

We use fractal analysis to calculate the protein concentration in a rotating magnetic assembly of microbeads of size 1 µm, which has optimized parameters of sedimentation, binding sites and magnetic volume. We utilize the original Forrest-Witten method, but due to the relatively small number of bead particles, which is of the order of 500, we use a large number of origins and also a large number of algorithm iterations. We find a value of the fractal dimension in the range 1.70-1.90, as a function of the thrombin concentration, which plays the role of binding the microbeads together. This is in good agreement with previous results from magnetorotation studies. The calculation of the fractal dimension using multiple points of reference can be used for any assembly with a relatively small number of particles.

Bead assembly magnetorotation as a signal transduction method for protein detection.

This paper demonstrates a proof-of-principle for a new signal transduction method for protein detection called Bead Assembly Magnetorotation (BAM). BAM is based on using the target protein to mediate the formation of aptamer-coated 1 µm magnetic beads into a bead assembly, formed at the bottom of a 1 µL hanging droplet. The size, shape and fractal dimension of this bead assembly all depend on the protein concentration. The protein concentration can be measured in two ways: by magnetorotation, in which the rotational period of the assembly correlates with the protein concentration, or by fractal analysis. Additionally, a microscope-free magnetorotation detection method is introduced, based on a simple laser apparatus built from standard laboratory components. In this paper, we chose to focus on the protein thrombin, a popular choice for proof-of-principle work in this field.

Influence of lightweight ambulatory oxygen on oxygen use and activity patterns of COPD patients receiving long-term oxygen therapy.

Lightweight ambulatory oxygen devices are provided on the assumptions that they enhance compliance and increase activity, but data to support these assumptions are lacking. We studied 22 patients with severe chronic obstructive pulmonary disease receiving long-term oxygen therapy (14 men, average age = 66.9 y, FEV(1) = 33.6%pred, PaO(2) at rest = 51.7 torr) who were using E-cylinders as their portable oxygen. Subjects were recruited at 5 sites and studied over a 2-week baseline period and for 6 months after randomizing them to either continuing to use 22-lb E-cylinders towed on a cart or to carrying 3.6-lb aluminum cylinders. Utilizing novel electronic devices, ambulatory and stationary oxygen use was monitored continuously over the 2 weeks prior to and the 6 months following randomization. Subjects wore tri-axial accelerometers to monitor physical activity during waking hours for 2-3 weeks prior to, and at 3 and 6 months after, randomization. Seventeen subjects completed the study. At baseline, subjects used 17.2 hours of stationary and 2.5 hours of ambulatory oxygen daily. At 6 months, ambulatory oxygen use was 1.4 ± 1.0 hrs in those randomized to E-cylinders and 1.9 ± 2.4 hrs in those using lightweight oxygen (P = NS). Activity monitoring revealed low activity levels prior to randomization and no significant increase over time in either group. In this group of severe chronic obstructive pulmonary disease patients, providing lightweight ambulatory oxygen did not increase either oxygen use or activity. Future efforts might focus on strategies to encourage oxygen use and enhance activity in this patient group. This trial is registered at ClinicalTrials.gov (NCT003257540).

Label-acquired magnetorotation as a signal transduction method for protein detection: aptamer-based detection of thrombin.

This paper presents a new signal transduction method, called label-acquired magnetorotation (LAM), for the measurement of the concentration of proteins in solution. We demonstrate the use of LAM to detect the protein thrombin using aptamers, with a limit of detection of 300 pM. LAM is modeled after a sandwich assay, with a 10 µm nonmagnetic "mother" sphere as the capture component and with 1 µm magnetic "daughter" beads as the labels. The protein-mediated attachment of daughter beads to the mother sphere forms a rotating sandwich complex. In a rotating magnetic field, the rotational frequency of a sandwich complex scales with the number of attached magnetic beads, which scales with the concentration of the protein present in solution. This paper represents the first instance of the detection of a protein using LAM.

Label-acquired magnetorotation for biosensing: An asynchronous rotation assay.

This paper presents a novel application of magnetic particles for biosensing, called label-acquired magnetorotation (LAM). This method is based on a combination of the traditional sandwich assay format with the asynchronous magnetic bead rotation (AMBR) method. In label-acquired magnetorotation, an analyte facilitates the binding of a magnetic label bead to a nonmagnetic solid phase sphere, forming a sandwich complex. The sandwich complex is then placed in a rotating magnetic field, where the rotational frequency of the sandwich complex is a function of the amount of analyte attached to the surface of the sphere. Here, we use streptavidin-coated beads and biotin-coated particles as analyte mimics, to be replaced by proteins and other biological targets in future work. We show this sensing method to have a dynamic range of two orders of magnitude.

Aptamers as affinity reagents in an integrated electrophoretic lab-on-a-chip platform.

Nucleic acid based affinity reagents (e.g., aptamers) offer several possible advantages over antibodies as specific recognition elements in biochemical assays. Besides offering improved cost and stability, aptamers are ideal for rapid electrophoretic analysis due to their low molecular weight and high negative charge. While aptamers have proven well-suited for affinity-shift electrophoretic analysis, demonstrating a fully integrated aptamer-based assay platform represents an important achievement toward low-cost point-of-care analysis, particularly for remote or resource poor settings where cost and ambient stability of reagents is a key consideration. Here we perform and evaluate the suitability of aptamer-based affinity assays for two clinically relevant target analytes (IgE using a known aptamer and NF-κB using a thio-modified aptamer) in an integrated electrophoretic gel-shift platform. Key steps of (i) mixing sample with aptamer, (ii) buffer exchange, and (iii) preconcentration of sample were successfully integrated on-chip upstream of a fluorescence-based gel-shift analysis step. This approach, utilizing a size-exclusion membrane optimized here for aptamer retention and preconcentration with sample, enables automated sample-to-answer for trace analytes in 10 min or less. We addressed notable nonspecific interference from serum proteins by adding similar nucleic acid competitors to suppress such interactions with the aptamer. Nanomolar sensitivities were demonstrated and integrated preconcentration of sample provides an important means of further improving detection sensitivities. Aptamers proved superior in many respects to antibody reagents, particularly with regard to speed and resolution of gel-shifts associated with specific binding to target.

Breath-by-breath quantification of progressive airflow limitation during exercise in COPD: a new method.

During heavy exercise in chronic obstructive pulmonary disease (COPD), dynamic airways compression leads to a progressive fall in intrabreath flow. This is manifested by concavity in the spontaneous expiratory flow-volume (SEFV) curve. We developed a method to quantify the SEFV curve configuration breath-by-breath during incremental exercise utilizing a computerized analysis. The flow signal was digitized at 100Hz. For each breath's SEFV curve, points of highest flow (V (max)) and end-expiration (V (EE)) were identified to define a rectangle's diagonal. Fractional area within the rectangle below the SEFV curve was defined as the "rectangular area ratio" (RAR); RAR <0.5 signifies concavity of the SEFV. To illustrate the utility of this method, time courses of RAR during incremental exercise in 12 healthy and 17 COPD individuals (FEV(1) %Pred.=39+/-12) were compared. SEFV in healthy individuals manifested progressively more convex SEFV curves throughout exercise (RAR=0.56+/-0.08 at rest and 0.61+/-0.05 at peak exercise), but became progressively more concave in COPD patients (RAR=0.52+/-0.08 at rest and 0.46+/-0.06 at peak exercise). In conclusion, breath-by-breath quantification of SEFV curve concavity describes progressive shape changes denoting expiratory flow limitation during incremental exercise in COPD patients. Further studies are warranted to establish whether this novel method can be a reliable indicator of expiratory flow limitation during exercise and to examine the relationship of RAR time course to the development of dynamic hyperinflation.

Methodology for using long-term accelerometry monitoring to describe daily activity patterns in COPD.

We sought to develop procedures for computerized analysis of long-term, high-resolution activity monitoring data that allow accurate assessment of the time course of activity levels suitable for use in chronic obstructive pulmonary disease (COPD) patients. Twenty-two COPD patients utilizing long-term oxygen recruited from 5 sites of the COPD Clinical Research Network wore a triaxial accelerometer (RT3, Stayhealthy, Monrovia, CA) during waking hours over a 14-day period. Computerized algorithms were composed allowing minute-by-minute activity data to be analyzed to determine, for each minute, whether the monitor was being worn. Temporal alignment allowed determination of average time course of activity level, expressed as average vector magnitude units (VMU, the vectorial sum of activity counts in three orthogonal directions) per minute, for each hour of the day. Mid-day activity was quantified as average VMU/minute between 10AM and 4PM for minutes the monitor was worn. Over the 14 day monitoring period, subjects wore the monitor an average of 11.4 +/- 3.0 hours x day(-1). During mid-day hours, subjects wore the monitor 76.3% of the time and generated an average activity level of 112 +/- 55 VMU x min(-1). Increase in precision of activity estimates with longer monitoring periods was demonstrated. This analysis scheme allows a detailed temporal pattern of activity to be defined from triaxial accelerometer recordings and has the potential to facilitate comparisons among subjects and between subject groups. This trial is registered at ClinicalTrials.gov (NCT00325754).